Bacterial pathogens utilize type III-VII translocation systems to inject a large cadre of protein effectors into the host cell leading to modulation of cellular processes and manifestation of disease. Although many injected bacterial effectors are targeted into host cellular membranes to attain biological function, the mechanisms of membrane targeting of injected bacterial effectors are not well understood. We have recently described a novel mechanism for anchoring an injected bacterial effector into host membranes through host-mediated farnesylation, which covalently links a protein to a 15-carbon farnesyl lipid moiety. Farnesylation of proteins is a conserved eukaryotic post-translational lipidation of conserved cysteine residue within the C-terminal teterapeptide CaaX motif of a protein. Farnesylation of hydrophilic proteins, such as Ras, enable the lipidated protein to be anchored into the lipid bi-layer of membranes to exhibit biological function. Within amoeba and human cells, the Legionnaires' disease bacterium, Legionella pneumophila (Lp), resides and proliferate within an ER-remodeled Legionella-containing vacuole (LCV), which evades lysosomal fusion. Remodeling the LCV within amoeba and human cells is dependent on the Dot/Icm type IV secretion system, which injects ~300 effectors into the host cell. We have shown that the Ankyrin B (AnkB) Dot/Icm-injected effector of Lp is anchored to the LCV membrane through host-mediated farnesylation of its CaaX motif, which is indispensable for Lp intracellular proliferation. Our preliminary data show that the host farnesylation enzymes are recruited to the cytosolic side of the LCV membrane, which is novel among intra-vacuolar pathogens. The cytosolic side of the LCV membrane is highly enriched with other farnesylated proteins, in addition to AnkB. In silico genomic analyses of Lp genomes have identified 6 Lp genes encoding novel proteins that harbor a C-terminal CaaX motif. The 6 encoded novel proteins are translocated into the host cell by the Dot/Icm system, and have been designated as prenylated effectors of Legionella (Pel). When the Pels are ectopically expressed in human cells, evidence suggests that the effectors associate with cellular membranes, but this membrane localization is perturbed upon: 1) inhibition of host farnesylation; or 2) substitution of the conserved cysteine residue (pel C-A) within the CaaX motif of the Pels. Importantly, a pelB null mutant and a pelB 502C-A substitution mutant exhibit similar severe defects in intracellular proliferation in macrophages. Therefore, our hypothesis is: The injected novel Pel effectors are farnesylated by the host cell farnesylation machinery that targets them to specific cellular membranes to exhibit their functions required for intracellular proliferation of Lp. To test the hypothesis, our specific aims are to determine: I) Host mediated farnesylation and membrane-anchoring of the Pel effectors; and 2) Role of the pel effectors and their farnesylation in the intracellular infection by Lp. The Significance of our studies: 1) Lp is a major cause of pneumonia; 2) The proposed studies will enhance our knowledge of Lp-host interaction through deciphering the role of novel translocated effectors in the intracellular infection; 3) Since our in silico genomic analyses show prevalence of the C-terminal CaaX motif in effectors of other pathogens with type III-VII translocated systems, our studies will stimulate investigations into a new paradigm in other pathogens; and 4) Our findings will contribute to our understanding the role of farnesylation in various human diseases, such as Ras-mediated malignancies. Innovation of the proposed studies come from: 1) the hypothesis; 2) hijacking the host farnesylation machinery by the pathogen-containing vacuole, which is highly enriched with farnesylated proteins; 3) Novelty of the 6 Pels Dot/Icm-translocated effectors, indicating novel functions in cellular modulations; 4) Our multidisciplinary approach of deciphering role of host-mediated lipidation of the Pel effectors combined with their biological functions in the intracellular infection by Lp; and 5) The major role for PelB and its C-terminal CaaX motif in the intracellular infection of human macrophages.